1. Field of the Invention
The present invention relates to methods for detecting the activity of an ion channel in a cell. The methods comprise providing a loading buffer solution to the cell, with the loading buffer comprises at least one environmentally sensitive thallium indicator (e g, luminescent dye) and chloride ions, and providing a stimulus buffer to the cell, wherein the stimulus buffer comprises thallium. Providing the stimulus buffer causes thallium influx into the cell through the ion channel. After providing the stimulus buffer, the luminescence (e.g., fluorescence) of the thallium indicator in the cell is detected, where the luminescence of the dye can change in the presence or absence of thallium.
2. Background of the Invention
High throughput screening (HTS) (e.g., HCS) of ion channels is currently limited by the efficiency of automated patch clamp instrumentation. The use of thallium influx as a surrogate indicator of potassium channel activity in clonal cell lines loaded with the calcium indicator BTC AM ester is now well-established (Weaver et al., Journal of Biomolecular Screening 2004; 9(8): 671-677). To date, the assays for monitoring ion channels use thallium (I), which selectively enters open potassium channels (Hille, J. Gen. Physiol. 1973; 61: 669-686) and binds to BTC, giving an optical readout of potassium channel activity. This method can be used to study the activation and/or inhibition of ion channels with drugs tested in HTS mode from a compound library.
The current methods, however, have significant drawbacks. Namely, thallium chloride is poorly soluble and will precipitate out of solution at a concentration of about 4.5 mM or greater. Thus, the buffers used for current methods must be essentially free of chloride to prevent TlCl from precipitating out of solution to generate data. The current methods, therefore, require additional steps of washing and removal of buffers in which cells are normally grown in culture (e.g., chloride containing buffers). Moreover, because chloride is absent in these assays, the assays can arguably be seen as not approximating physiological conditions.